Detecting Individual Proteins and Their Surface Charge Variations in Solution by the Potentiometric Nanoimpact Method

Label-free detection and analysis of proteins in their natural form and their dynamic interactions with substrates at the single-molecule level are important for both fundamental studies and various applications. Herein, we demonstrate a simple potentiometric method to achieve this goal by detecting the native charge of protein in solution by utilizing the principle of single-entity electrochemistry techniques. When a charged protein moves near the vicinity of a floating carbon nanoelectrode connected to a high-impedance voltage meter, the distinct local electrostatic potential changes induced by the transient collision event of protein, also called the “nanoimpact” event, can be captured by the nanoelectrode as a potential probe. This potentiometric method is highly sensitive for charged proteins, and low-molecular-weight proteins less than 10 kDa can be detected in low-salt-concentration electrolytes. By analyzing the shape and magnitude of the recorded time-resolved potential change and its time derivative, we can reveal the charge and motion of the protein in the nonspecific protein–surface interaction event. The charge polarity variations of the proteins at different pH values were also successfully probed. Compared with synthetic spherical nanoparticles, the statistical analysis of many single-molecule nanoimpact events revealed a large variation in the recorded transient potential signals, which may be attributed to the intrinsic protein dynamics and surface charge heterogeneity, as suggested by the finite element method and molecular dynamic simulations

Additional file 1 of GRP75-driven, cell-cycle-dependent macropinocytosis of Tat/pDNA-Ca2+ nanoparticles underlies distinct gene therapy effect in ovarian cancer

Additional file 1. Table S1. Mean size and zeta potential of Tat/pDNA-Ca2+ nanoparticles

MOESM1 of Arf6-driven endocytic recycling of CD147 determines HCC malignant phenotypes

Additional file 1: Figure S1. CD147 expression and stable knock-down in liver cancer cells. Figure S2. Flow cytometry analysis of CD147 level on liver cancer cell surface. Figure S3. Arf6-mediated CD147 recycling promotes Huh7 and HepG2 cell adhesion to ECM. Figure S4. Arf6-KD impaired the cell-cell aggregation of Huh7 and HepG2 cells. Arf6-perturbed cells were reseeded on agar for static culture, and cell aggregation clusters were evaluated. Figure S5. Morphometric analyses of Arf6-perturbed liver cancer cells. Figure S6. ARF6-specific GEFs and GAPs expressed in liver cancer patients. Box plots depict the expression level difference between liver cancer (T) and normal tissues (N). Figure S7. Co-expression network analysis of the Arf6-CD147 gene pair. Figure S8. Pair-wise correlation analysis for the expression (IHC staining) levels of CD147, Arf6, Rac1 and ARNO in primary HCC tissues. Table S1. Clinicopathological features of HCC patients and association with co-expression of CD147, Arf6, ARNO, and Rac1

Additional file 2 of GRP75-driven, cell-cycle-dependent macropinocytosis of Tat/pDNA-Ca2+ nanoparticles underlies distinct gene therapy effect in ovarian cancer

Additional file 2: Fig. S1. Granularity and electric potential analysis of Tat/pGL3 and Tat/pGL3-Ca2+ particles. Fig. S2. High-concentration, long-term treatment with Tat/pGL3-Ca2+ nanoparticles triggers necrotic apoptosis. Fig. S3. Stability characteristics of Tat/pDNA-Ca2+ nanoparticles in culture media or mice serum. Fig. S4. Unpackaging of Tat/pDNA complexes or Tat/pDNA-Ca2+ nanoparticles by heparin displacement of pDNA. Fig. S5. Tat/pGL3-Ca2+ nanoparticles mainly use macropinocytosis for uptake. Fig. S6. EIPA treatment inhibited the uptake and expression of Tat/pGL3-Ca2+ nanoparticles. Fig. S7. Tat/pDNA-Ca2+ nanoparticles do not interfere with sub-phase distribution of cell-cycle. Fig. S8. Construction of recombinant lentiviral plasmids for GRP75 over-expression (A) and knock-down (B). Fig. S9. Highly expression or phosphorylated activation of GRP75 promotes centrosome duplication in Cos7 cells, and GRP75 mainly localizes in duplicated centrosome. Fig. S10. Highly expression or phosphorylated activation of GRP75 promotes itself and Mps1 co-translocating to centrosome in Cos7 cells. Fig. S11. GRP75 and Mps1 co-localized with r-tubulin only in duplicating centrosome. Fig. S12. Quantification of apoptotic cells in ovarian tumor with different treatments. Fig. S13. H&E staining of hearts, livers, spleens, lungs, kidneys and tumor tissues from mice with different treatments

Anticancer Drug Doxorubicin Spontaneously Reacts with GTP and dGTP

Here, we reported a spontaneous reaction between anticancer drug doxorubicin and GTP or dGTP. Incubation of doxorubicin with GTP or dGTP at 37 °C or above yields a covalent product: the doxorubicin-GTP or -dGTP conjugate where a covalent bond is formed between the C14 position of doxorubicin and the 2-amino group of guanine. Density functional theory calculations show the feasibility of this spontaneous reaction. Fluorescence imaging studies demonstrate that the doxorubicin-GTP and -dGTP conjugates cannot enter nuclei although they rapidly accumulate in human SK-OV-3 and NCI/ADR-RES cells. Consequently, the doxorubicin-GTP and -dGTP conjugates are less cytotoxic than doxorubicin. We also demonstrate that doxorubicin binds to ATP, GTP, and other nucleotides with a dissociation constant (Kd) in the sub-millimolar range. Since human cells contain millimolar levels of ATP and GTP, these results suggest that doxorubicin may target ATP and GTP, energy molecules that support essential processes in living organisms

Exploring the Conformational and Binding Dynamics of HMGA2·DNA Complexes Using Trapped Ion Mobility Spectrometry–Mass Spectrometry

The mammalian high mobility group protein AT-hook 2 (HMGA2) is an intrinsically disordered DNA-binding protein expressed during embryogenesis. In the present work, the conformational and binding dynamics of HMGA2 and HMGA2 in complex with a 22-nt (DNA22) and a 50-nt (DNA50) AT-rich DNA hairpin were investigated using trapped ion mobility spectrometry–mass spectrometry (TIMS–MS) under native starting solvent conditions (e.g., 100 mM aqueous NH4Ac) and collision-induced unfolding/dissociation (CIU/CID) as well as solution fluorescence anisotropy to assess the role of the DNA ligand when binding to the HMGA2 protein. CIU-TIMS–CID-MS/MS experiments showed a significant reduction of the conformational space and charge-state distribution accompanied by an energy stability increase of the native HMGA2 upon DNA binding. Fluorescence anisotropy experiments and CIU-TIMS–CID-MS/MS demonstrated for the first time that HMGA2 binds with high affinity to the minor groove of AT-rich DNA oligomers and with lower affinity to the major groove of AT-rich DNA oligomers (minor groove occupied by a minor groove binder Hoechst 33258). The HMGA2·DNA22 complex (18.2 kDa) 1:1 and 1:2 stoichiometry suggests that two of the AT-hook sites are accessible for DNA binding, while the other AT-hook site is probably coordinated by the C-terminal motif peptide (CTMP). The HMGA2 transition from disordered to ordered upon DNA binding is driven by the interaction of the three basic AT-hook residues with the minor and/or major grooves of AT-rich DNA oligomers